Cell culture and chemicals
The human liver carcinoma cell lines HepG2 and Huh7 were purchased from Shanghai Bank of Cell Culture, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). All cells were cultured in the RPMI1640 medium (Gibco, USA) supplemented with 10% FBS (fetal bovine serum, Gibco) and 1% penicillin/streptomycin (GIBCO) at 37 °C in a humidified incubator with 5 % CO2. Caspase-3 inhibitor (Q-DEVD-OPh), caspase-8 inhibitor (Z-IETD-FMK), caspase-9 inhibitor (Z-LEHD-FMK) and pan-caspase inhibitor (z.VAD-FMK) were obtained from Abcam (USA). All other chemicals used were obtained from Sigma-Aldrich (USA).
MTT assay
HepG2 and Huh-7 cells were seeded into 96-well plates at the density of 5000 cells/well. After treated with various doses of protopine under various conditions, cell viabilities were measured using the MTT assay (Sigma) as described previously [11].
Wound healing assay
Cellular migration was measured using the wound healing assay. Cells were seeded into the 24-well plate at the density of 1 × 104 cells/well. 24 hours later, cells were cultured to 90% confluence, a scratch was created by a sterile tip on the monolayer cells and cells were treated with various doses of protopine. 24 h later, the width of wound was measured under an inverted microscope (Olympus, Japan) at × 100 magnification and normalized to initial distance at 0 h.
Invasion assay
Transwell assay was performed to measure cellular invasion. Cells were suspended in serum-free medium containing various doses of protopine at the density of about 400 cells/μl and seeded into the upper chambers of transwell which was coated with the Matrigel (BD Biosciences, USA), 500 μl of full culture medium with 10% FBS was placed into the bottom chamber as the chemoattractant. After culture for 48 h, the cells remaining in the top chambers were removed and the cells in the bottom chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of invaded cells were counted under the inverted microscope (Olympus, Japan) at × 100 magnification.
Cellular apoptosis assay
After being treated with various doses of protopine for 24 h, 1 × 105 cells were collected and staining with 500 μl Annexin V binding buffer with 5 μl of Annexin V-FITC at room temperature for 0.5 h and avoid the light. Then cellular apoptosis was measured using the flow cytometer (BD Biosciences, USA).
Relative caspase activities assay
The relative activities of caspase-3, caspase-8 and caspase-9 were measured by the caspase-3, caspase-8 and caspase-9 multiplex activity assay kit (Abcam, USA) according to the manufacturer’s guide. In brief, cells were treated with various doses of protopine for 24 h, and cellular lysates were collected. Equal amount of cellular lysates (10 μl) was added into 96-well plates and 80 μl of reaction buffer containing various caspase substrates. After incubated at 37 °C for 4 h, caspase activities were measured at the absorbance at 450 nm.
ROS measurement
Levels of ROS were measured by staining cells with 2’7’-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich) according to the manufacturer’s guide. After treated with various doses of protopine for 24 h, then cells were incubated with 10 mM DCFH-DA for 0.5 h to avoid light, and fluorescence was detected by flow cytometry (BD Bioscience, USA).
MDA, SOD, LDH and GPX assays
The levels of MDA, SOD, LDH and GPX were measured by the Lipid Peroxidation (MDA) Assay kit, SOD activity assay kit, LDH assay kit and Glutathione Peroxidase (GPX) Assay Kit, respectively according to the manufacturer’s guide. All kits were obtained from the Abcam.
RNA sequencing analysis
Cells were treated with or without protopine for 24 h and total RNA was then extracted for RNA-seq in triplicate. RNA-seq was performed and analyzed by GeneChem Ltd (Shanghai, China). Various signalling pathways were investigated and the significant differentially expressed genes (P<0.05) were classified into corresponding signalling pathways.
Western blotting assay
Cells were harvested and lysed with RIPA buffer after transfection. The protein concentrations were assayed a Bradford protein assay kit (Beyotime). Equal amounts of protein (20 μg) were separated by 12% SDS-PAGE and transferred to PVDF membranes (Merck-Millipore Bioscience) at 100 V for 1 hour. The blots were blocked with 5% skimmed milk at room temperature for 1 h, followed by incubation with different primary antibodies at 4 °C overnight. All antibodies were obtained from the Cellular Signalling Technologies (USA). Signals were visualized using an enhanced chemiluminescence (ECL; Pierce, USA).
In vivo study
Male BALB/c mice at 6–8 weeks were chosen and randomly divided into four groups (n= 5/group). Cells were implanted (107 cells/ml) subcutaneously into the right frontal axils of the mice. When the tumour volume reached about 100 mm3, the mice were treated with an intravenous injection of saline (control) or various doses of protopine. Tumor volume was measured once every 3 days and calculated by the formula: volume = (width2 × length)/2. At the end of the treatment, the tumours were resected and the weight of tumours were measured. Then the tumours protein was extracted using the One-Step animal tissue active protein extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s guide. All animal experiments followed ethical standards and all protocols were approved by the animal using committee of Zhejiang University.
Statistical Analysis
Data were analysed using SPSS 12.0 software (Chicago, IL, USA). All experiments were conducted at least three times. The results are expressed as the mean ± SD. Difference between groups was compared using One-way ANOVA followed Tukey’s post-hoc test. A value of p<0.05 was considered significantly different.